Synthesis and characterization of a PAMAM-OH derivative containing an acid-labile β-thiopropionate bond for gene delivery

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Abstract

The present report describes the synthesis of a hydroxyl terminal PAMAM dendrimer (PAMAM-OH) derivative (PAMSPF). The hydroxyls of PAMAM-OH were attached to S-Methyl-l-cysteine (SMLC) via an acid-labile ester bond, named as β-thiopropionate bond, followed by modification with folic acid (FA) through a polyethylene glycol (PEG) linker. The degrees of attachment of SMLC and FA to the PAMAM-OH backbone were 83.9% and 12.8%, respectively. PAMSPF could condense DNA to form spherical nanoparticles with particle sizes of ∼200 nm and remain stable in the presence of heparin and nuclease. The β-thiopropionate bond in PAMSPF was hydrolyzed completely and the DNA release rate was 95.8 ± 3.3% after incubation under mildly acidic conditions at 37 °C for 3 h. PAMSPF/DNA was less cytotoxic to KB and HepG2 cells and exhibited a higher gene transfection efficiency than native PAMAM/DNA. The uptake assays showed that PAMSPF/DNA entered KB cells within 0.5 h through folate receptor-mediated endocytosis and escaped from endosomes within 2 h. In addition, PAMSPF/DNA displayed long circulation time along with excellent targeting of tumor sites in vivo. These findings demonstrate that PAMSPF is an excellent carrier for safe and effective gene delivery.

Introduction

Recently, gene therapy has been restricted mainly because of the absence of safe and effective gene delivery carriers. Although viral vectors have many merits, potential risks limit their clinical application. As alternatives to viral vectors, various kinds of nonviral gene delivery carriers, including polycations, lipids and peptides, have been developed and studied because of their advantages, such as lac of immunogenic effects, size flexibility, ease of handling and effective incorporation of therapeutic genes (De Smedt et al., 2000, Gao et al., 2016). Among these, polyamidoamine dendrimer (PAMAM) is an attractive option due to its well-defined structure and multivalent functional groups that can be further modified for gene delivery applications. In endosome vesicles, the secondary and tertiary amines of the PAMAM dendrimer undergo marked changes in protonation to promote endosomal escape, fulfilling the so-called “proton sponge hypothesis” (Eichman et al., 2000, Esfand and Tomalia, 2001).

However, PAMAM also has a few shortcomings, such as limited transfection efficiency and high cytotoxicity (Nam et al., 2009). It is well known that the surface functional groups largely dictate the cytotoxic effects of polycations. The hydroxyl terminal PAMAM dendrimer (PAMAM-OH) has a similar structure to PAMAM except for the replacement of surface amine groups with hydroxyl groups. PAMAM-OH has been reported to be relatively nontoxic due to the absence of any surface positive charges (Patil et al., 2009). Moreover, PAMAM-OH still fulfills the “proton spong hypothesis” and is able to escape from endosomes due to its interior secondary and tertiary amines (Lee et al., 2003, Nam et al., 2008).

Many studies have shown that increased DNA release can promote transfection efficiency by the degradation of carriers (Nguyen et al., 2009, Ou et al., 2008). The transfection activities of biodegradable PGBELA/DNA and PBELA/DNA are significantly different, with mean values of 6.4 × 105 and 3.3 × 105 RLU/mg protein, respectively (Chen et al., 2012). Early computational models of non-viral gene delivery using an integrative system approach have shown that the efficient release of DNA from its carrier is a critical step in the transfection process (Dinh et al., 2007). A first-order mass-action model predicts a dependence of transgene expression on the rate of DNA release, which has been validated using in vitro transfection data (Varga et al., 2001). Thus, it seems that carriers have to degrade in order to release gene payloads rapidly after endocytosis (Cornelis et al., 2002, Zabner et al., 1995). If this does not occur, the DNA might not undergo the process of transcription and eventually be lost or diluted by processes including exocytosis and mitosis (Grigsby and Leong, 2010).

Taking advantage of the pH differential between serum (pH 7.4) and endosome organelles (pH 5.0) (Binauld et al., 2012, Tannock and Rotin, 1989), the strategy for the design of degradable polymer carriers containing acid-labile linkages (Guk et al., 2013, Kim et al., 2005, Yuan et al., 2010) has been widely used to achieve a high transfection efficiency. The backbones of these polymers generally remain intact under basic conditions, and readily degrade in endosome organelles due to the cleavage of the acid-labile linkages. As polyplexes circulate throughout the body they slowly release their DNA payload. Once internalized in endosomes, polyplexes are degraded and release the encapsulated DNA efficiently. In the present study our interest is focused on a particular ester bond named β-thiopropionate, which has been reported to be easily hydrolyzed at a mildly acidic pH in endosomes (Dan et al., 2011, Oishi et al., 2005, Oishi et al., 2003). The lone pair electrons of the sulfide atom at the β-position of the carbonyl may facilitate the formation of a four-membered ring, which can be attacked by water molecules and, thus, the β-thiopropionate is more sensitive to acid than the ordinary ester bond.

Based on the results of previous studies, this report focuses on the preparation of a biodegradable PAMAM-OH derivative (PAMSPF) as a gene delivery carrier. PAMAM-OH was conjugated with S-Methyl-L-cysteine (SMLC) via a β-thiopropionate bond followed by modification with folic acid (FA) through a polyethylene glycol (PEG) linker. The PAMSPF/DNA-polyplex (PAMSPF/DNA) displayed a high capacity for condensing DNA to protect it under physiological conditions and release it under acidic conditions due to the degradation of the β-thiopropionate bond. The main degradation products, PAMAM-OH and SMLC, had no or only very weak positive charges in the cytoplasm. Also, the isoelectric point of SMLC was lower than the cytoplasmic pH (Kurkdjian and Guern, 1989) and, thus, SMLC exhibited a negative charge, which allowed it to repel the negatively charged DNA due to the absence of an ionic interaction. As a result, PAMSPF/DNA can unpack DNA through two pathways: degradation of the carrier and the repelling force between the degradation products and DNA. In addition, the low cytotoxicity is obtained due to the lack of positive charges. Furthermore, PAMSPF/DNA displays a long circulation time along with a great ability to target to tumor sites in vivo. A schematic diagram depicting the formation and the (ex-) intracellular trafficking of the PAMSPF/DNA is shown in Fig. 1.

Section snippets

Materials

Hydroxyl terminal PAMAM dendrimer (PAMAM-OH, G4.0), amino terminal PAMAM dendrimer (PAMAM, G4.0) and a hetero-functional PEG derivative (NH2-PEG-COOH) with an average molecular weight of 2 kDa were obtained from Sigma-Aldrich (St. Louis, MO, USA). N-Methyl-l-cysteine (SMLC), folic acid (FA), dimethyl sulfoxide (DMSO), 1-ethyl-(3-3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 4-dimethylaminopyridine (DMAP), N-hydroxysuccinimide ester (NHS), triethylamine (TEA), trifluoroacetic acid

Synthesis of PAMAM-OH derivatives

In this study, SMLC was combined with the hydroxyls of PAMAM-OH using a condensing agent, EDC/DMAP (Navath et al., 2010). Firstly, we should determine whether there was any some unreacted SMLC remained in the product and HPLC analysis was used. As shown in Fig. 3A, the retention time of free SMLC was 5 min and there was not the significant peak of SMLC in the PAMS. As a result, PAMS was purified. Similar results were obtained for PAMSP and PAMSPF (data not shown). The structure of the

Conclusion

In the present study, a multifunctional gene delivery system, PAMSPF/DNA, was prepared. The degradation of PAMSPF/DNA under acidic conditions could increase both the DNA release and transfection efficiency. Moreover, PAMSPF/DNA displayed lower cytotoxicity than native PAMAM/DNA, exhibited FRs-mediated cellular uptake and facilitated endosomal escape from tumor cells. In addition, PAMSPF/DNA exhibited an excellent long-termcirculating effect and targeting ability in tumor-bearing mice. We

Acknowledgments

The authors are grateful to the Natural Science Foundation Committee of China for financial support (NO. 81173004 and NO. 81202483).

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